PrimPol is required for replication reinitiation after mtDNA damage

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved September 1, 2017 (received for review April 2, 2017)
October 9, 2017
114 (43) 11398-11403

Significance

Failure to maintain mtDNA integrity can lead to a wide variety of neuromuscular disorders. Despite its central role in the development of these disorders, many mechanistic details of mtDNA maintenance are still unclear. In the present work, we have studied the role of PrimPol, an unusual primase-polymerase, in mammalian mtDNA maintenance. We report here that PrimPol is specifically required for replication reinitiation after DNA damage. PrimPol synthesizes DNA primers on an ssDNA template, which can be elongated by the mitochondrial replicative polymerase γ, a solution to reprime replication beyond DNA lesions and to facilitate lagging-strand replication. Our findings show that PrimPol has biological relevance for mtDNA maintenance.

Abstract

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

Continue Reading

Acknowledgments

We thank Sandra Chocrón and Maria Martínez-Jiménez for valuable PrimPol-related discussions and work and Mr. Craig Michell (University of Eastern Finland) for language editing. This work was supported by the Jane & Aatos Erkko (JAE) Foundation (R.T.-M. and J.L.O.P.), the Finnish Academy (S.G.), the Wallenberg Foundation (S.W., J.M.E.F., and A.P.), Olle Engkvist Byggmästare Foundation (G.S.), Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (G.C.), and Spanish Ministry of Economy and Competitiveness Grant BFU2015-65880-P (to L.B.).

Supporting Information

Supporting Information (PDF)

References

1
L Wan, et al., hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity. EMBO Rep 14, 1104–1112 (2013).
2
S García-Gómez, et al., PrimPol, an archaic primase/polymerase operating in human cells. Mol Cell 52, 541–553 (2013).
3
TA Guilliam, BA Keen, NC Brissett, AJ Doherty, Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes. Nucleic Acids Res 43, 6651–6664 (2015).
4
LM Iyer, EV Koonin, DD Leipe, L Aravind, Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: Structural insights and new members. Nucleic Acids Res 33, 3875–3896 (2005).
5
AA Bocquier, et al., Archaeal primase: Bridging the gap between RNA and DNA polymerases. Curr Biol 11, 452–456 (2001).
6
E Matsui, et al., Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii. Biochemistry 42, 14968–14976 (2003).
7
MK Zafar, A Ketkar, MF Lodeiro, CE Cameron, RL Eoff, Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2′-deoxyguanosine. Biochemistry 53, 6584–6594 (2014).
8
J Bianchi, et al., PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol Cell 52, 566–573 (2013).
9
S Mourón, et al., Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nat Struct Mol Biol 20, 1383–1389 (2013).
10
MI Martínez-Jiménez, et al., Alternative solutions and new scenarios for translesion DNA synthesis by human PrimPol. DNA Repair (Amst) 29, 127–138 (2015).
11
K Kobayashi, et al., Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle 15, 1997–2008 (2016).
12
D Schiavone, et al., PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells. Mol Cell 61, 161–169 (2016).
13
L Kazak, A Reyes, IJ Holt, Minimizing the damage: Repair pathways keep mitochondrial DNA intact. Nat Rev Mol Cell Biol 13, 659–671 (2012).
14
DA Clayton, JN Doda, EC Friedberg, The absence of a pyrimidine dimer repair mechanism in mammalian mitochondria. Proc Natl Acad Sci USA 71, 2777–2781 (1974).
15
KJ Krishnan, et al., What causes mitochondrial DNA deletions in human cells? Nat Genet 40, 275–279 (2008).
16
AF Phillips, et al., Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion. MolCell 65, 527–538.e526 (2017).
17
S Goffart, et al., Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling. Hum Mol Genet 18, 328–340 (2009).
18
S Wanrooij, S Goffart, JL Pohjoismäki, T Yasukawa, JN Spelbrink, Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes. Nucleic Acids Res 35, 3238–3251 (2007).
19
JL Martin, CE Brown, N Matthews-Davis, JE Reardon, Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis. Antimicrob Agents Chemother 38, 2743–2749 (1994).
20
G Stojkovič, et al., Oxidative DNA damage stalls the human mitochondrial replisome. Sci Rep 6, 28942 (2016).
21
R Torregrosa-Muñumer, S Goffart, JA Haikonen, JL Pohjoismäki, Low doses of ultraviolet radiation and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates, fork regression, and replication initiation shift. Mol Biol Cell 26, 4197–4208 (2015).
22
JL Pohjoismäki, S Goffart, Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA. BioEssays 33, 290–299 (2011).
23
A Reyes, et al., Mitochondrial DNA replication proceeds via a ‘bootlace’ mechanism involving the incorporation of processed transcripts. Nucleic Acids Res 41, 5837–5850 (2013).
24
JL Pohjoismäki, et al., Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397, 1144–1155 (2010).
25
TA Brown, C Cecconi, AN Tkachuk, C Bustamante, DA Clayton, Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev 19, 2466–2476 (2005).
26
J Miralles Fusté, et al., In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication. PLoS Genet 10, e1004832 (2014).
27
T Yasukawa, et al., Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J 25, 5358–5371 (2006).
28
XH Pham, et al., Conserved sequence box II directs transcription termination and primer formation in mitochondria. J Biol Chem 281, 24647–24652 (2006).
29
JM Fusté, et al., Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication. Mol Cell 37, 67–78 (2010).
30
SE Lim, WC Copeland, Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma. J Biol Chem 276, 23616–23623 (2001).
31
NA Cavanaugh, RD Kuchta, Initiation of new DNA strands by the herpes simplex virus-1 primase-helicase complex and either herpes DNA polymerase or human DNA polymerase alpha. J Biol Chem 284, 1523–1532 (2009).
32
DN Frick, CC Richardson, DNA primases. Annu Rev Biochem 70, 39–80 (2001).
33
EL Huttlin, et al., The BioPlex network: A systematic exploration of the human interactome. Cell 162, 425–440 (2015).
34
LJ Bailey, J Bianchi, N Hégarat, H Hochegger, AJ Doherty, PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage. Cell Cycle 15, 908–918 (2016).
35
JL Pohjoismäki, S Goffart, The role of mitochondria in cardiac development and protection. Free Radic Biol Med 106, 345–354 (2017).
36
AK Hyvärinen, et al., The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA. Nucleic Acids Res 35, 6458–6474 (2007).
37
JL Pohjoismäki, et al., Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells. Nucleic Acids Res 34, 5815–5828 (2006).
38
JL Alexander, TL Orr-Weaver, Replication fork instability and the consequences of fork collisions from rereplication. Genes Dev 30, 2241–2252 (2016).
39
JL Pohjoismäki, S Goffart, JN Spelbrink, Replication stalling by catalytically impaired Twinkle induces mitochondrial DNA rearrangements in cultured cells. Mitochondrion 11, 630–634 (2011).
40
JL Pohjoismäki, et al., Human heart mitochondrial DNA is organized in complex catenated networks containing abundant four-way junctions and replication forks. J Biol Chem 284, 21446–21457 (2009).
41
M Bowmaker, et al., Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J Biol Chem 278, 50961–50969 (2003).
42
S Goffart, H Spelbrink, Inducible expression in human cells, purification, and in vitro assays for the mitochondrial DNA helicase Twinkle. Methods Mol Biol 554, 103–119 (2009).
43
MY Yang, et al., Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication. Cell 111, 495–505 (2002).
44
N Atanassova, et al., Sequence-specific stalling of DNA polymerase γ and the effects of mutations causing progressive ophthalmoplegia. Hum Mol Genet 20, 1212–1223 (2011).
45
E Scotto-Lavino, G Du, MA Frohman, 5′ end cDNA amplification using classic RACE. Nat Protoc 1, 2555–2562 (2006).
46
BJ Brewer, WL Fangman, The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471 (1987).
47
JE Kolesar, CY Wang, YV Taguchi, SH Chou, BA Kaufman, Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome. Nucleic Acids Res 41, e58 (2013).
48
JA Korhonen, XH Pham, M Pellegrini, M Falkenberg, Reconstitution of a minimal mtDNA replisome in vitro. EMBO J 23, 2423–2429 (2004).
49
JM Gerhold, A Aun, T Sedman, P Jõers, J Sedman, Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication. Mol Cell 39, 851–861 (2010).
50
S Crews, D Ojala, J Posakony, J Nishiguchi, G Attardi, Nucleotide sequence of a region of human mitochondrial DNA containing the precisely identified origin of replication. Nature 277, 192–198 (1979).
51
B Xu, DA Clayton, RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: An implication for RNA-DNA hybrids serving as primers. EMBO J 15, 3135–3143 (1996).
52
J Fish, N Raule, G Attardi, Discovery of a major D-loop replication origin reveals two modes of human mtDNA synthesis. Science 306, 2098–2101 (2004).
53
Z Livneh, et al., High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA. DNA Repair (Amst) 44, 59–67 (2016).
54
V Pagès, Single-strand gap repair involves both RecF and RecBCD pathways. Curr Genet 62, 519–521 (2016).
55
BAI Payne, et al., Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet 43, 806–810 (2011).
56
AM Furda, AS Bess, JN Meyer, B Van Houten, Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative PCR. Methods Mol Biol 920, 111–132 (2012).
57
C Frezza, S Cipolat, L Scorrano, Organelle isolation: Functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2, 287–295 (2007).
58
KS Dimmer, et al., LETM1, deleted in Wolf-Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability. Hum Mol Genet 17, 201–214 (2008).

Information & Authors

Information

Published in

The cover image for PNAS Vol.114; No.43
Proceedings of the National Academy of Sciences
Vol. 114 | No. 43
October 24, 2017
PubMed: 29073063

Classifications

Submission history

Published online: October 9, 2017
Published in issue: October 24, 2017

Keywords

  1. DNA repair
  2. fork rescue
  3. mtDNA damage
  4. mtDNA replication

Acknowledgments

We thank Sandra Chocrón and Maria Martínez-Jiménez for valuable PrimPol-related discussions and work and Mr. Craig Michell (University of Eastern Finland) for language editing. This work was supported by the Jane & Aatos Erkko (JAE) Foundation (R.T.-M. and J.L.O.P.), the Finnish Academy (S.G.), the Wallenberg Foundation (S.W., J.M.E.F., and A.P.), Olle Engkvist Byggmästare Foundation (G.S.), Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (G.C.), and Spanish Ministry of Economy and Competitiveness Grant BFU2015-65880-P (to L.B.).

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Rubén Torregrosa-Muñumer1
Department of Environmental and Biological Sciences, University of Eastern Finland, 80101 Joensuu, Finland;
Josefin M. E. Forslund1
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
Steffi Goffart
Department of Environmental and Biological Sciences, University of Eastern Finland, 80101 Joensuu, Finland;
Annika Pfeiffer
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
Gorazd Stojkovič
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
Gustavo Carvalho
Centro de Biologia Molecular Severo Ochoa, E-28049 Madrid, Spain
Natalie Al-Furoukh
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
Luis Blanco
Centro de Biologia Molecular Severo Ochoa, E-28049 Madrid, Spain
Sjoerd Wanrooij3,2 [email protected]
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden;
Department of Environmental and Biological Sciences, University of Eastern Finland, 80101 Joensuu, Finland;

Notes

3
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: S.G., L.B., S.W., and J.L.O.P. designed research; R.T.-M., J.M.E.F., S.G., A.P., G.S., G.C., and N.A.-F. performed research; S.G., L.B., and S.W. contributed new reagents/analytic tools; R.T.-M., J.M.E.F., A.P., G.C., and J.L.O.P. analyzed data; and R.T.-M., J.M.E.F., S.G., G.S., L.B., S.W., and J.L.O.P. wrote the paper.
1
R.T.-M. and J.M.E.F. contributed equally to this work.
2
S.W. and J.L.O.P. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

Metrics & Citations

Metrics

Note: The article usage is presented with a three- to four-day delay and will update daily once available. Due to ths delay, usage data will not appear immediately following publication. Citation information is sourced from Crossref Cited-by service.


Citation statements

Altmetrics

Citations

Export the article citation data by selecting a format from the list below and clicking Export.

Cited by

    Loading...

    View Options

    View options

    PDF format

    Download this article as a PDF file

    DOWNLOAD PDF

    Login options

    Check if you have access through your login credentials or your institution to get full access on this article.

    Personal login Institutional Login

    Recommend to a librarian

    Recommend PNAS to a Librarian

    Purchase options

    Purchase this article to access the full text.

    Single Article Purchase

    PrimPol is required for replication reinitiation after mtDNA damage
    Proceedings of the National Academy of Sciences
    • Vol. 114
    • No. 43
    • pp. 11259-E9182

    Figures

    Tables

    Media

    Share

    Share

    Share article link

    Share on social media